注射用重组人干扰素β1b规模化分离纯化工艺的建立及其产品质量的检定
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摘要
目的建立注射用重组人干扰素β1b(recombinant human interferonβ1b,rh IFNβ1b)规模化纯化工艺,并对该工艺制备产品的质量进行检定。方法采用高压均质机对rh IFNβ1b/BL21菌体进行破碎,离心提取包涵体,经有机抽提及酸化沉淀获得粗制rhIFNβ1b;通过Sephacryl S-200和Sephadex G-25层析纯化获得rhIFNβ1b原液,连续生产3批。将原液稀释,加入保护剂,经分装、冻干制备成品。同时对原液及成品的质量进行检定。结果 3批rhIFNβ1b原液蛋白产量可达6~7 g,平均回收率可达15.1%,平均效价为1.1×10~7 IU/mg,HPLC纯度和SDS-PAGE纯度均超过97%,一级序列和二级结构具有良好的一致性。每批rhIFNβ1b原液可制备出20 000~30 000支成品,且各项检定项目均合格。结论建立了rhIFNβ1b规模化分离纯化工艺,制品的产量、纯度及活性均较高,符合rhIFNβ1b工业化生产的要求。
Objective To establish a process for large-scale purification of recombinant human interferon(IFN) β1b for injection and control the quality of product. Methods The recombinant E. coli was homogenized, and inclusion bodies were collected by centrifugation, with which crude rh IFNβ1b was prepared by organic extraction and acid precipitation,and purified by Sephacryl S-200 and Sephadex G-25 chromatography to obtain the bulk. Three consecutive batches of bulk were prepared, which were diluted, added with stabilizer, filled and lyophilized to prepare the final product. Control tests were performed on bulk and final product. Results The yield of rh IFN-β1b protein of three batches of bulk were 6 ~ 7 g,while the mean recovery rate was 15. 1%, the mean titer was 1. 1 × 107 IU/mg, both the purities were more than 97% by both HPLC and SDS-PAGE, and the primary sequence was consistent with the secondary structure. About 20 000 ~30 000 containers of final product were prepared with a single batch of bulk, of which all the quality indexes met the relevant requirements. Conclusion A process for large-scale purification of rh IFN-β1b was established, and the product with high yield, purity and activity was obtained, which met the requirements for industrial production of rh IFN-β1b.
Objective To establish a process for large-scale purification of recombinant human interferon(IFN) β1b for injection and control the quality of product. Methods The recombinant E. coli was homogenized, and inclusion bodies were collected by centrifugation, with which crude rh IFNβ1b was prepared by organic extraction and acid precipitation,and purified by Sephacryl S-200 and Sephadex G-25 chromatography to obtain the bulk. Three consecutive batches of bulk were prepared, which were diluted, added with stabilizer, filled and lyophilized to prepare the final product. Control tests were performed on bulk and final product. Results The yield of rh IFN-β1b protein of three batches of bulk were 6 ~ 7 g,while the mean recovery rate was 15. 1%, the mean titer was 1. 1 × 107 IU/mg, both the purities were more than 97% by both HPLC and SDS-PAGE, and the primary sequence was consistent with the secondary structure. About 20 000 ~30 000 containers of final product were prepared with a single batch of bulk, of which all the quality indexes met the relevant requirements. Conclusion A process for large-scale purification of rh IFN-β1b was established, and the product with high yield, purity and activity was obtained, which met the requirements for industrial production of rh IFN-β1b.
引文
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[19]BOUBLIK M,MOSCHERA J A,WEI C,et al.Conformation and activity of recombinant human fibroblast interferon-β[J].J Interferon Res,1990,10(2):213-219.
[2]BANDARI D S,STERNAMAN D,CHAN T,et al.Evaluating risks,costs,and benefits of new and emerging therapies to optimize outcomes in multiple sclerosis[J].J Manag Care Pharm,2012,18(9):1-17.
[3]MARZINIAK M,MEUTH S.Current perspectives on interferon Beta-1b for the treatment of multiple sclerosis[J].Adv Ther,2014,31(9):915-931.
[4]ABDOLVAHAB M H,FAZELI A,RADMALEKSHALI M,et al.An albumin-free formulation for Escherichia coli-derived interferon beta-1b with decreased immunogenicity in immune tolerant mice[J].J Interferon Cytokine Res,2016,36(3):192-203.
[5]KALLURI S R,GRUMMEL V,HRACSKO Z,et al.Interferon-beta specific T cells are associated with the development of neutralizing antibodies in interferon-beta treated multiple sclerosis patients[J].J Autoimmun,2017,pii:S0896-8411(17)30587-5.doi:10.1016/j.jaut.2017.10.003.
[6]MCKAY F C,HOE E,PAMELL G,et al.IL7Rαexpression and upregulation by IFNβin dendritic cell subsets is haplotype-dependent[J].PLo S One,2013,8(10):e77508.doi:10.1371/journal.pone.0077508.
[7]TOLLEY K,HUTCHINSON M,YOU X,et al.A network meta-analysis of efficacy and evaluation of safety of subcutaneous pegylated interferon beta-1a versus other injectable therapies for the treatment of relapsing-remitting multiple sclerosis[J].PLo S One,2015,10(6):e0127960.doi:10.1371/journal.pone.0127960.
[8]YANG X,ZHANG X,FU M L,et al.Targeting the tumor microenvironment with interferon-βbridges innate and adaptive immune responses[J].Cancer Cell,2014,25(1):37-48.
[9]TSUGE M,UCHIDA T,HIRAGA N,et al.Development of a novel site-specific pegylated interferon beta for antiviral therapy of chronic hepatitis B virus[J].Antimicrob Agents Chemo-ther,2017,61(6):pii:e00183-17.doi:10.1128/AAC.00183-17.
[10]ANTONELLI G,SCAGNOLARI C,VICENZI E,et al.Treatment of SARS with human interferons[J].Lancet,2003,362(9390):1158.
[11]MCCARTHY S D,MAJCHRZAK-KITA B,RACINE T,et al.A rapid screening assay identifies monotherapy with interferon-βand combination therapies with nucleoside analogs as effective inhibitors of Ebola virus[J].PLo S Negl Trop Dis,2016,10(1):e0004364.doi:10.1371/journal.pntd.0004364.
[12]JOHNSTON J,SO T Y.First-line disease-modifying therapies in paediatric multiple sclerosis:a comprehensive overview[J].Drugs,2012,72(9):1195-1211.
[13]ZHANG Z L,LIU J Y,YANG Y K,et al.Preparation of recombinant human interferonβSer17 and its inhibitory effect SARS virus[J].Chin J Biologicals,2005,18(6):501-503.(in Chinese)张振龙,刘建源,杨云凯,等.重组人干扰素βSer17的制备及其对SARS病毒的抑制作用[J].中国生物制品学杂志,2005,18(6):501-503.
[14]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Repubic of China(VolⅢ)[S].Beijing:China Med Sci Press,2015:3404-3523.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:3404-3523.
[15]HU M Z,LI W H,YAN Q X,et al.Relationship of interferon-αbetween its conformation and anti-viral property analyzed by circular dichroism and flow cutometry[J].Chin J Biotech,2015,31(11):1651-1659.(in Chinese)胡茂志,李文华,严秋香,等.利用圆二色谱和流式细胞术分析干扰素-α的构象与其抗病毒活性的相关性[J].生物工程学报,2015,31(11):1651-1659.
[16]LIN L S,YAMAMOTO R,DRUMMOND R J.Purification of recombinant human interferonβexpressed in Escherichia coli[J].Methods Enzymol,1986,119(4):183-192.
[17]ZHANG Z L,LIU J Y,YANG Y K,et al.Purification and quality control of recombinant human IFN-β1b for injection[J].Chin J New Drugs,2005,14(7):887-890.(in Chinese)张振龙,刘建源,杨云凯,等.注射用重组人干扰素β1b的分离纯化及质量研究[J].中国新药杂志,2005,14(7):887-890.
[18]YANG Y K,MA Y,SUN Y J,et al.Determination of interferonβ1b content in recombinant human interferonβ1b for inje-ction by reversed phase high performance liquid cheomatography[J].Chin J Biologicals,2015,28(10):1053-1056.(in Chinese)杨云凯,马昱,孙玉杰,等.反相高效液相色谱法测定注射用重组人干扰素β1 b中干扰素β1b的含量[J].中国生物制品学杂志,2015,28(10):1053-1056.
[19]BOUBLIK M,MOSCHERA J A,WEI C,et al.Conformation and activity of recombinant human fibroblast interferon-β[J].J Interferon Res,1990,10(2):213-219.